Mitochondrial Isozyme Research Articles

Pyruvate Dehydrogenase Kinase 4 Deficiency Increases Tumorigenesis in a Murine Model of Bladder Cancer.

Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial isozyme in the PDK family (PDK1-4) partially responsible for phosphorylation of pyruvate dehydrogenase (PDH). Phosphorylation of PDH is thought to result in a pro-proliferative shift in metabolism that sustains growth of cancer cells. Previous data from our lab indicate the pan-PDK inhibitor dichloroacetate (DCA) or acute genetic knockdown of PDK4 blocks proliferation of bladder cancer (BCa) cells. The goal of this study was to determine the role of PDK4 in an in vivo BCa model, with the hypothesis that genetic depletion of PDK4 would impair formation of BCa. PDK4-/- or WT animals were exposed to N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 16 weeks, and tumors were allowed to develop for up to 7 additional weeks. PDK4-/- mice had significantly larger tumors at later time points. When animals were treated with cisplatin, PDK4-/- animals still had larger tumors than WT mice. PDK4 expression was assessed in human tissue and in mice. WT mice lost expression of PDK4 as tumors became muscle-invasive. Similar results were observed in human samples, wherein tumors had less expression of PDK4 than benign tissue. In summary, PDK4 has a complex, multifunctional role in BCa and may represent an underrecognized tumor suppressor.

Unsupervised Learning for Feature Representation Using Spatial Distribution of Amino Acids in Aldehyde Dehydrogenase (ALDH2) Protein Sequences

Aldehyde dehydrogenase 2 (ALDH2) enzyme is required for alcohol detoxification. ALDH2 belongs to the aldehyde dehydrogenase family, the most important oxidative pathway of alcohol digestion. Two main liver isoforms of aldehyde dehydrogenase are cytosolic and mitochondrial. Approximately 50% of East Asians have ALDH2 deficiency (inactive mitochondrial isozyme), with lysine (K) for glutamate (E) substitution at position 487 (E487K). ALDH2 deficiency is also known as Alcohol Flushing Syndrome or Asian Glow. For people with an ALDH2 deficiency, their face turns red after drinking alcohol, and they are more susceptible to various diseases than ALDH2-normal people. This study performed a machine learning analysis of ALDH2 sequences of thirteen other species by comparing them with the human ALDH2 sequence. Based on the various quantitative metrics (physicochemical properties, secondary structure, Hurst exponent, Shannon entropy, and fractal dimension), these fourteen species were clustered into four clusters using the unsupervised machine learning (K-means clustering) algorithm. We also analyze these species using hierarchical clustering (agglomerative clustering) and draw the phylogenetic trees. The results show that Homo sapiens is more closely related to the Bos taurus and Sus scrofa species. Our experimental results suggest that the testing for discovering medicines may be done on these species before being tested in humans to alleviate the impacts of ALDH2 deficiency.

Repression of Mitochondrial Citrate Synthase Genes by Aluminum Stress in Roots of Secale cereale and Brachypodium distachyon.

Aluminum (Al) toxicity in acid soils influences plant development and yield. Almost 50% of arable land is acidic. Plants have evolved a variety of tolerance mechanisms for Al. In response to the presence of Al, various species exudate citrate from their roots. Rye (Secale cereale L.) secretes both citrate and malate, making it one of the most Al-tolerant cereal crops. However, no research has been done on the role of the mitochondrial citrate synthase (mCS) gene in Al-induced stress in the rye. We have isolated an mCS gene, encoding a mitochondrial CS isozyme, in two S. cereale cultivars (Al-tolerant cv. Ailés and Al-sensitive inbred rye line Riodeva; ScCS4 gene) and in two Brachypodium distachyon lines (Al-tolerant ABR8 line and Al-sensitive ABR1 line; BdCS4 gene). Both mCS4 genes have 19 exons and 18 introns. The ScCS4 gene was located on the 6RL rye chromosome arm. Phylogenetic studies using cDNA and protein sequences have shown that the ScCS4 gene and their ScCS protein are orthologous to mCS genes and CS proteins of different Poaceae plants. Expression studies of the ScCS4 and BdSC4 genes show that the amount of their corresponding mRNAs in the roots is higher than that in the leaves and that the amounts of mRNAs in plants treated and not treated with Al were higher in the Al-tolerant lines than that in the Al-sensitive lines of both species. In addition, the levels of ScCS4 and BdCS4 mRNAs were reduced in response to Al (repressive behavior) in the roots of the tolerant and sensitive lines of S. cereale and B. distachyon.

ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ

To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. MethodsA compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. ResultsCross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. ConclusionWe conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.

Mitochondrial MTHFD isozymes display distinct expression, regulation, and association with cancer

Mitochondrial folate metabolism is central to the generation of nucleotides, fuelling methylation reactions, and redox homeostasis. Uniquely among the reactions of the mitochondrial folate pathway, the key step of the oxidation of 5,10-methylene-tetrahydrofolate (CH2-THF) can be catalysed by two isozymes, MTHFD2 and MTHFD2L. The MTHFD2 enzyme has recently received considerable attention as an oncogenic enzyme upregulated in several tumour types, which is additionally required by cancer cells in vitro and in vivo. However, much less is currently known about MTHFD2L and its expression in cancer. In this study, we examine and compare the expression and regulation of the two mitochondrial MTHFD isozymes in normal human and cancer cells. We found that normal and cancer cells express both enzymes, although MTHFD2 has a much higher baseline expression. Unlike MTHFD2, the MTHFD2L isozyme does not show an association with proliferation and growth factor stimulation. In addition, we did not find evidence of a compensatory increase of MTHFD2L following suppression of its isozyme. This study supports that MTHFD2L is unlikely to have an important function in increased proliferation or cancer. Furthermore, therapeutic strategies aiming to block the mitochondrial folate pathway in cancer should focus on MTHFD2, with MTHFD2L being unlikely to be involved in the development of chemoresistance to targeting of its mitochondrial isozyme.

The Effect of Different Hormones and Antibiotics on Activity of AST Enzyme and its Isozymes in Wistar Rats

Background: One of the valuable tests for diagnosis of cardiovascular and liver diseases is measuring of AST activity. One of the main enzymes of transaminases group is aspartate aminotransferase. Previous Studies have shown that some alteration may occur in mitochondria function as the result of different disease or taking different medication; these changes in mitochondrial and cytosolic AST isozymes can be the sign of disorders. According to the role of steroid hormone in induction of its effects on protein synthesis genes, this study is conducted to shed some light on mechanisms and the interference of steroid hormones and antibiotics.Materials, Methods & Results: In this study, male Wistar rats were injected intramuscularly with Testosterone, progesterone and estradiol; while tetracycline and streptomycin injections were intraperitoneal. Testosterone, progesterone and estradiol injections were carried out in a short-term (15 days) and long-term (45 days) periods. Steroid hormones were dissolved in sesame in a way that by each injection, 0.2 mL sesame oil (containing specific amount of hormone) was injected to the rat. Control group was kept in the same condition except that their sesame oil injection contained no hormone. Tetracycline and Streptomycin injection was carried out for 5 days at 7 am and pm, at 50 mg/kg dosage intraperitoneally. In short- and long-term periods, rats were divided into four groups of 6-member. The concentrations were the same in the periods and 0.2 mL sesame oil was injected intramuscularly. 1 mg testosterone, 12 mg progesterone and 0.2 mg estradiol were intramuscularly injected to rats in group 2, 3 and 4, respectively [10]. Rats were divided into 9 six-member groups as follows: Group 1: intraperitoneal injection of 0.2 mL physiological serum; group 2: injection of 1 mg testosterone; group 3: injection of 1 mg testosterone + 50 mg/kg streptomycin; group 4: injection of 1 mg testosterone + 50 mg/kg tetracycline; group 5: injection of 0.2 mg estradiol; group 6: injection of 0.2 mg estradiol + 50 mg/kg streptomycin; group 7: injection of 0.2 mg estradiol + 50 mg/kg tetracycline; group 8: injection 50 mg/kg streptomycin; and group 9: injection of 50 mg/kg tetracycline. Serum concentration of AST enzyme was measured at the end of each period and the data were compared by SPSS software. all three steroid hormones had no significant impact on AST activity in short term. However, a significant effect was observed in long term in mean AST activities of the 4 groups. The group injected by testosterone exhibited 9% increases in comparison with the control group. Antibiotic-administrated groups showed lower activities as compared with hormone-injected groups. Steroid hormones and testosterone can enhance AST activity, in short-term and long-term, respectively by induction of protein enzyme. The second test confirmed this theory as the antibiotics decreased the AST activity enhancement by testosterone.Discussion: Based on the present study, steroid hormones can enhance the aspartate aminotransferase activity; and antibiotics can decrease the level of this liver enzyme by inhibition of polypeptide synthesis on related genes. This reaction could be due to interference of hormones and antibiotics effect which hinders the hormone effect along with the drug to activate the protein synthesis process.

N-Sulfamoylphenyl- and N-sulfamoylphenyl-N-thiazolyl-β-alanines and their derivatives as inhibitors of human carbonic anhydrases

A series of N-substituted and N,N-disubstituted β-amino acids and their derivatives bearing benzenesulfonamide moiety were designed and synthesized in search of compounds that would be high-affinity and selective inhibitors of human carbonic anhydrases (CA). There are 12 catalytically active human CA isoforms, the cytosolic CA I, CA II, CA III, CA VII, and CA XIII, secreted CA VI, the mitochondrial CA VA and CA VB, membrane-associated CA IV, and transmembrane CA IX, CA XII, and CA XIV. The di-bromo meta-substituted compounds exhibited low nanomolar dissociation constants and over 10-fold selectivity for mitochondrial isozyme CA VB, implicated in diseases of the central nervous system and obesity. These compounds can be used for further development as inhibitors of significant binding affinity and selectivity towards CA VB isozyme.

Активність та вміст ізозимів аспартатамінотрансферази в репродуктивних органах корів

Activity and isozyme content of aspartate transaminase in cow reproductive organs at different physiological states and ovarian hypofunction were studied. It was registered, that activity of aspartate transaminase (AST) in ovarian tissue and endometrium at corpus luteum state is 132.0 ± 8.23 and 172.9 ± 8.70 μm/min×mg of protein, lower on 26.3 and 5.5% at follicle growth and at early corpus luteum is 66.5 ± 2.10 and 147.1 ± 13.26 μm/min×mg of protein. Lowest AST activity is registered at hypofunction state (61.8 ± 4.57 і 146.1 ± 9.33 μm/min×mg of protein). In cow reproductive organ tissues and antral fluid from ovarian follicles was registered two bands of proteins, that have AST activity. In reproductive organs main part is mitochondrial isozyme (АSТ2; 78.8–97.7%) in antral fluid – cytosole (АSТ1; 72.8–88.2%). In ovarian and uterus tissues was established high enzyme activity at late corpus luteum, that characterizes intensive aspartate conversion to oxaloacetate and possible utilization of substeates to maintain energetic and sytnthetic needs in cow reproductive organs. Antral fluid of big follicles from ovarian at follicle growth and at state of late corpus luteum characterizes by high activity and content of AST, that points on follicle cell growth – granulose and oocyte. In ovarian tissue and endometrium at hypofunction lower activity and isozyme content point on substrate deficiency for transamination and violation of physiological functions.

Cardiac Mitochondrial Respiratory Dysfunction and Tissue Damage in Chronic Hyperglycemia Correlate with Reduced Aldehyde Dehydrogenase-2 Activity.

Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial isozyme of the heart involved in the metabolism of toxic aldehydes produced from oxidative stress. We hypothesized that hyperglycemia-mediated decrease in ALDH2 activity may impair mitochondrial respiration and ultimately result in cardiac damage. A single dose (65 mg/kg; i.p.) streptozotocin injection to rats resulted in hyperglycemia with blood glucose levels of 443 ± 9 mg/dl versus 121 ± 7 mg/dl in control animals, p<0.0001, N = 7–11. After 6 months of diabetes mellitus (DM) induction, the rats were sacrificed after recording the functionality of their hearts. Increase in the cardiomyocyte cross sectional area (446 ± 32 μm2 Vs 221 ± 10 μm2; p<0.0001) indicated cardiac hypertrophy in DM rats. Both diastolic and systolic dysfunctions were observed with DM rats compared to controls. Most importantly, myocardial ALDH2 activity and levels were reduced, and immunostaining for 4HNE protein adducts was increased in DM hearts compared to controls. The mitochondrial oxygen consumption rate (OCR), an index of mitochondrial respiration, was decreased in mitochondria isolated from DM hearts compared to controls (p<0.0001). Furthermore, the rate of mitochondrial respiration and the increase in carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)-induced maximal respiration were also decreased with chronic hyperglycemia. Chronic hyperglycemia reduced mitochondrial OXPHOS proteins. Reduced ALDH2 activity was correlated with mitochondrial dysfunction, pathological remodeling and cardiac dysfunction, respectively. Our results suggest that chronic hyperglycemia reduces ALDH2 activity, leading to mitochondrial respiratory dysfunction and consequently cardiac damage and dysfunction.

Activities of cytochrome c oxidase and mitochondrial lactate dehydrogenase isozymes and Cox1, Cox2, Cox4, and Cox6 gene subunit expression in cold adaptation of Salmo trutta L.

Characteristic changes in some parameters of white muscle mitochondria (mitochondrial volume, activity of cytochrome c oxidase (COX, EC 1.9.3.1) and the level of Cox1, Cox2, Cox4, and Cox6 subunit gene expression and activity and kinetic characteristics of mitochondrial lactate dehydrogenase isozymes (mtLDH, EC 1.1.1.27)) in adaptation to seasonal decrease in temperature from 16 to 6°C of one-year juvenile brown trout Salmo trutta L. from rivers of Lake Onega basin were investigated. A 1.5-fold increase in the activity of COX, and the increase in the levels of gene expression of both Cox4 and Cox6 nuclear subunits and increased activity of mitochondrial LDH isozymes, which have a low affinity towards lactate, has been shown. The possible role of nuclear and mitochondrial subunits of cytochrome c oxidase in improving the efficiency of the enzyme in molecule biogenesis and further modulation of its activity, as well as regulation of pyruvate formation to maintain the required rate of oxidative phosphorylation in mitochondria under low temperature were discussed.

Enzyme Activities of Muscle Tissue Lysates can be Unexpectedly Affected by Protease Inhibitor Cocktails

Previous studies in our lab have shown that the protective use of certain protease inhibitor cocktails with skeletal muscle tissue lysates can decrease protein expression on PAGE (polyacrylamide gel electrophoresis). The purpose of the present study was to determine whether inclusion of cocktails in two different rat skeletal muscle (biceps femoris and rectus abdominis) tissue lysates could affect enzyme activities of endogenous non-proteases. After lysates were incubated with cocktails at 37°C for one hour, they were run on non-denaturing polyacrylamide gel electrophoresis (native-PAGE) and assayed for enzyme activities. Results indicate that acetyl-cholinesterase (AChE) activities of lysates incubated with cocktails were significantly diminished; in contrast, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activities of lysates incubated with cocktails were unaffected. When muscle lysates were challenged with high trypsin concentrations (0.14%), enzyme activities of MDH (mitochondrial isozyme) and LDH were inhibited; however, the inclusion of certain cocktails rescued activities. Enzyme activities in cardiac and small intestinal muscle tissue lysates treated with cocktails appear to show a similar pattern. In conclusion, it is suggested that the inclusion of cocktails with tissue lysates should be carefully evaluated when measuring tissue enzyme activity. (Support from MSMC Research Funding and Pace Scholarly Research)

Expression and distribution of thiol-regulating enzyme glutaredoxin 2 (GRX2) in porcine ocular tissues

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase's distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea, iris, and the lens. No Grx2 mRNA, protein or enzyme activity could be found in the vitreous humor. The results indicate that Grx2 level was higher in eye tissues rich in vasculature and mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is also consistent with their known sensitivity to oxidative stress.

Abstract 57: Impaired Aldehyde Dehydrogenase 2 Activity Contributes To Inhibition Of Mitochondrial Respiration In Hyperglycemic Rats

Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial isozyme of the heart involved in the metabolism of toxic aldehydes produced from oxidative stress. A decrease in ALDH2 levels and activity was reported in the hearts of experimental hyperglycemia along with an increase in 4-hydroxy-2-nonenal (4HNE). 4HNE is produced in the mitochondria upon lipid peroxidation and known to form adduct with mitochondrial proteins. We hypothesize that reduced ALDH2 activity impairs mitochondrial respiration due to 4HNE adducts formation, ultimately resulting in cardiac damage in hyperglycemia. A single dose (65 mg/kg; i.p.) of streptozotocin in rats resulted in hyperglycemia with a blood glucose level of 443 ± 9 mg/dl versus 121 ± 7 mg/dl in control animals, p&lt;0.0001. N=7-11 in both groups. After 6 months of hyperglycemia, heart function was recorded and the rats were sacrificed. Increases in cardiomyocyte cross sectional area (446 ± 32 μm 2 Vs 221 ± 10 μm 2 ; p&lt;0.0001) indicated cardiac hypertrophy in diabetic rats. Both diastolic and systolic dysfunctions were recorded with diabetic rats compared to controls. For instance, % fractional shortening was lower in diabetic group versus control; (p&lt;0.0001). Most importantly, myocardial ALDH2 activity and levels were reduced and immunostaining for 4HNE protein adducts was increased in diabetic hearts compared to controls. The mitochondrial oxygen consumption rate (OCR), an index of mitochondrial respiration, was decreased in mitochondria isolated from diabetic hearts compared to controls (p&lt;0.0001). To check the direct effect of 4HNE and ALDH2 inhibition, we treated H9C2 cardiomyocytes with 4HNE and disufiram, an ALDH inhibitor, respectively, which reduced mitochondrial respiration (p&lt;0.0001) and cell viability (p&lt;0.0001) along with reduced ALDH2 activity and increased 4HNE adducts formation. We conclude that chronic hyperglycemia-induced 4HNE accumulation due to low ALDH2 activity in the heart leads to defective mitochondrial respiration and cardiac damage and dysfunction.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis

Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice. The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using (2)H and (13)C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes. PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms. We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver.

(R)-/(S)-10-Camphorsulfonyl-substituted aromatic/heterocyclic sulfonamides selectively inhibit mitochondrial over cytosolic carbonic anhydrases

A series of aromatic and heterocyclic sulfonamides incorporating R- and S-camphorsulfonyl moieties were synthesized and investigated for the inhibition of several mammalian isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The new sulfonamides selectively inhibited the mitochondrial isozymes hCA VA and VB (h=human isoform) over the cytosolic, off-target ones hCA I and II, with inhibition constants in the low nanomolar range. The chirality and position of the groups substituting the sulfonamide scaffold greatly influenced CA inhibitory properties. These compounds are excellent leads for designing isoform-selective enzyme inhibitors targeting mitochondrial CAs involved in lipogenesis and obesity.

Compartmentalization of Mammalian Folate-Mediated One-Carbon Metabolism

The recognition that mitochondria participate in folate-mediated one-carbon metabolism grew out of pioneering work beginning in the 1950s from the laboratories of D.M. Greenberg, C.G. Mackenzie, and G. Kikuchi. These studies revealed mitochondria as the site of oxidation of one-carbon donors such as serine, glycine, sarcosine, and dimethylglycine. Subsequent work from these laboratories and others demonstrated the participation of folate coenzymes and folate-dependent enzymes in these mitochondrial processes. Biochemical and molecular genetic approaches in the 1980s and 1990s identified many of the enzymes involved and revealed an interdependence of cytoplasmic and mitochondrial one-carbon metabolism. These studies led to the development of a model of eukaryotic one-carbon metabolism that comprises parallel cytosolic and mitochondrial pathways, connected by one-carbon donors such as serine, glycine, and formate. Sequencing of the human and other mammalian genomes has facilitated identification of the enzymes that participate in this intercompartmental one-carbon metabolism, and animal models are beginning to clarify the roles of the cytoplasmic and mitochondrial isozymes of these enzymes. Identifying the mitochondrial transporters for the one-carbon donors and elucidating how flux through these pathways is controlled are two areas ripe for exploration.

Carbonic anhydrase inhibitors. Identification of selective inhibitors of the human mitochondrial isozymes VA and VB over the cytosolic isozymes I and II from a natural product-based phenolic library

We have investigated the enzyme inhibition characteristics of a natural product (NP)-based phenolic library against a panel of human carbonic anhydrases (hCAs, EC 4.2.1.1) which included hCAs I and II (cytosolic) and hCA VA/VB (mitochondrial isoforms). Most of these compounds were weak, micromolar inhibitors of the two cytosolic hCAs ( K Is >10 μM) but showed good hCA VA/VB inhibitory activity with inhibition constants in the range of 70–125 nM. The selectivity ratios for inhibiting the mitochondrial over the cytosolic isoforms for these phenol derivatives were in the range of 120–3800, making them the most isoform-selective compounds for inhibiting hCA VA/VB known to date. The CA VA/VB enzymes are involved in biosynthetic processes such as gluconeogenesis, lipogenesis and ureagenesis, and no pharmacological inhibitors with good selectivity are currently available. Thus the NP inhibitors identified during these studies are excellent leads for obtaining even more effective compounds that selectively target mitochondrial hCAs, and also have the potential to be used as tools for understanding the physiological processes that are regulated by the two mitochondrial CA isoforms.

Novel organometallic cationic ruthenium(II) pentamethylcyclopentadienyl benzenesulfonamide complexes targeted to inhibit carbonic anhydrase

Cationic ruthenium(II) pentamethylcyclopentadienyl benzenesulfonamide sandwich complexes have been synthesized and screened for enzymatic inhibition of the physiologically dominant carbonic anhydrase (CA) isozymes: human CA I and II, mitochondrial isozymes VA and VB, and the cancer-associated isozyme IX. The complexes demonstrated weaker binding to CAs compared with typical aromatic sulfonamides, inhibiting the enzyme at high nanomolar concentrations. An in vitro cytotoxic evaluation of the complexes was also undertaken against a range of tumorigenic cell lines and a healthy human cell line. Complexes inhibited the growth of cancerous cells at low micromolar concentrations while expressing lower levels of toxicity towards the normal human cell line. Factors influencing the synthesis, cytotoxicity, and enzyme affinity for this series of organometallic complexes are discussed.